Human N-terminal pro-brain natriuretic peptide (NT-proBNP) ELISA kit instruction manual
September 26 00:12:43, 2025
**Human N-terminal pro-brain natriuretic peptide (NT-proBNP) ELISA Kit – Instructions for Use**
This kit is intended for research use only and is designed to quantitatively determine the concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) in human serum, plasma, and other biological fluids. The method employed is a double-antibody sandwich ELISA, which ensures high specificity and accuracy.
**Principle of Operation:**
The assay involves immobilizing a specific antibody against NT-proBNP onto the surface of microtiter plate wells. After incubation with the sample, the target antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming a complex. Following washing steps, the TMB substrate is introduced, and the reaction is stopped with an acidic solution. The resulting color change, measured at 450 nm, is directly proportional to the NT-proBNP concentration in the sample.
**Kit Components:**
- 48-well or 96-well microplate (coated with primary antibody)
- Standard: 90 ng/L (0.5 mL × 1 bottle)
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme-labeled reagent: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Substrate A & B: 3 mL × 1 bottle each
- Wash Buffer (20×): 20 mL × 20 times (for 48-well), 20 mL × 30 times (for 96-well)
- Sealing film: 2 pieces
- Storage: 2–8°C
**Sample Preparation:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant; mix well and centrifuge similarly.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection, or lyse cells via freeze-thaw cycles if intracellular components are to be measured.
- **Tissue Homogenate:** Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant.
**Procedure Summary:**
1. Prepare standard dilutions in duplicate.
2. Add sample and standards to designated wells.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with wash buffer.
5. Add HRP-conjugated antibody and incubate again.
6. Add TMB substrate and develop for 15 minutes.
7. Stop the reaction with stop solution.
8. Measure absorbance at 450 nm using a microplate reader.
**Notes and Precautions:**
- Ensure all reagents are brought to room temperature before use.
- Avoid repeated freeze-thaw cycles of samples.
- Do not use NaN3-containing samples, as it may inhibit HRP activity.
- Always run a standard curve and include blank controls.
- Keep all components sealed when not in use to prevent contamination.
- Discard all waste materials as biohazardous.
**Calculation:**
Plot the OD values of the standards against their concentrations to create a standard curve. Use linear regression to calculate the unknown sample concentration, adjusting for any dilution factor.
**Performance Characteristics:**
- Correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 2 ng/L – 80 ng/L
**Storage and Shelf Life:**
- Store the kit at 2–8°C.
- Valid for 6 months from the date of manufacture.
Always follow the manufacturer’s instructions carefully and ensure proper training and equipment are available for accurate results.
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